Understanding Next Generation Sequencing in Forensic Science

A Q&A with Kelly Elkins and Cynthia Zeller, authors of Next Generation Sequencing in Forensic Science: A Primer

This article appears in the September-October 2021 issue of Evidence Technology Magazine.
You can view that full issue here.

NEXT-GENERATION SEQUENCING (NGS) technology has rapidly gained attention as the future of forensic DNA analysis. Since the first high-profile application of NGS in 2018 to the Golden State Killer case, a plethora of other cases have made use of the technology to solve cases where mixtures and degraded samples would usually present issues for traditional DNA analysis.

Dr. Kelly Elkins

To help provide a quick overview of what makes NGS so powerful, what it means to existing forensic laboratory workflows, and where it’s going in the future, we conducted a question-and-answer session with Dr. Kelly Elkins and Cynthia Zeller, authors of the book Next Generation Sequencing in Forensic Science: A Primer. Elkins and Zeller are both associate professors of chemistry at Towson University in Maryland and co-directors of the Towson University Human Remains DNA Identification Laboratory.

To get started, we asked the authors a little about their backgrounds:

What drew you to forensic science?

Elkins: For as long as I can remember, I loved science, learning how machines work, and making observations outside on everything from how grasshoppers jump to how sand flows. I majored in biology and chemistry in college. I was drawn to forensic science as it brought together my interests in history and mysteries to DNA and chemical analysis. I like how the forensic science field applies cutting-edge science to solve real problems and cases to make our communities safer.

Zeller: Like Kelly, I also majored in biology and chemistry in college. I spent years studying the role of carbohydrates on cell-cell interactions before embarking on a career as a DNA analyst with the Maryland State Police Division of Forensic Sciences, and finally as an associate professor at Towson University. At the Maryland State Police, I was able to use my skills to provide data that was used to ensure justice for the victims of crime, as well as the wrongly accused.

What compelled you to write a book about next-generation sequencing?

Elkins: Towson University invested in NGS technology for forensic applications in 2018 and we taught our first class, Next Generation Sequencing in Forensics, in the spring semester of 2019. In preparing for our class and developing the curriculum, we drew from the primary literature, manufacturer resources, webinars, and conference posters and oral presentations. There was no ready-to-use course materials or book we could use or draw from. We wrote this book to share our reviews of the literature and reference lists and insights from using NGS to make it easier for other faculty and lab directors to incorporate NGS to their courses and workflows. We’ve taught the class for the past three years and are happy to finally offer our students a book to go with the course.

What are some challenges with traditional forensic DNA analysis?

Elkins: Traditional forensic analysis is limited by how many loci can be probed in one analysis. As a result, evaluating autosomal STRs, X-STRs, Y-STRs, and SNPs each requires input DNA to run one or more analysis. This approach is slow and requires sufficient input DNA for each analysis, which is not available in many cases. Additionally, traditional DNA methods cannot differentiate identical twins. They analyze lengths of short tandem repeat DNA loci rather than sequences and miss sequence variation between samples.

How does NGS differ from traditional sequencing?

Elkins: Many of the NGS methods were developed from the principles of traditional Sanger sequencing in which each DNA nucleotide is labeled with a different dye that fluoresces at a unique wavelength. Whereas each sample and locus was sequenced sequentially in traditional sequencing, in NGS many samples and loci are sequenced in parallel at the same time, leading to sequence data for millions to billions of targets in a run.

What were the first applications of NGS to forensic casework?

Elkins: The first case that drew wide attention from the forensic community was the application of NGS and investigative genetic genealogy to the Golden State Killer case in 2018.

What types of samples / cases is NGS best suited for?

Elkins: NGS can be used to analyze any evidence sample but is currently mostly being used for cold cases and missing persons cases. NGS is an excellent tool for cases in which a low quantity of DNA has been recovered from evidence, as forensic scientists can simultaneously analyze a large panel of autosomal STR, Y-STR, X-STR, and SNP sites. Not only is the DNA able to be used for all of these analyses simultaneously, NGS is also more sensitive than traditional methods.

Describe the benefits of NGS when applied to mixtures.

Elkins: NGS is a sequencing technique. Sequencing STRs leads to more information and the potential for differentiating samples, even if they have the same number of STR repeats at a site. This is incredibly useful in differentiating mixtures. NGS is also more sensitive than traditional methods, so forensic scientists are more likely to capture data from a sample using NGS than traditional STR typing methods.

How effective is NGS with degraded samples?

Elkins: Degraded samples often lead to partial profiles using traditional methods. NGS typically leads to a more complete DNA profile. Identity-informative SNPs that are analyzed in NGS panels but not routinely in traditional methods can provide information that can be integral to a case even when a full STR profile is not obtained. SNP sites are smaller since they focus on the identity of a single base in the genome and are much more likely to be typed in DNA analysis.

How much new equipment is required to add NGS capabilities to a lab’s workflow?

Elkins: Only one new instrument is required to add NGS to a lab’s workflow and that is the sequencer itself. Certainly, automating preparation steps with a robot can free scientists from these steps to focus their time on data analysis and reporting steps that are a part of any DNA analysis. Tools including fragment analyzers can provide scientists insight into the likelihood of their samples producing full or partial profiles with NGS.

Is training difficult?

Elkins: Manufacturers have made the kits as easy to use and protocols as simple to follow as traditional DNA typing kits. Using and maintaining the NGS instrument requires much less time and effort as compared to capillary electrophoresis instruments. There are fewer moving parts, and the consumables are pre-packaged in a block that just needs to be inserted into the instrument along with the sequencing flow cell.

Is NGS faster?

Elkins: NGS is much faster than traditional methods if a lab is interested in collecting autosomal STR, X-STR, Y-STR and SNP data on case samples.

What hurdles does a lab face if it wants to adopt NGS?

Elkins: The lab needs to acquire an NGS instrument. Thereafter, it needs to fund training for its scientists, and they need to validate the new instrument and kits they will use just as when they introduce any new technology and methods to their lab workflows. Other NGS-specific considerations include determining how and where the data will be stored.

How much more data storage does NGS require compared to traditional analysis?

Elkins: NGS produces much more data than traditional DNA typing methods. It analyzes more loci and leads to sequence data, not just length data for STRs. As most of us know from using digital cameras, the photo quality and resolution continues to increase but also our hard drives and cloud storage have had to expand to accommodate them. Many NGS instruments take photographs of the fluorescence produced as each base is added in the sequence in the sequencing step. These colors in these images are analyzed to determine which nucleotide base was added. Hundreds of images may be collected in each sequencing run. Although the images comprise the largest files, there are also interpretation and analysis files associated with each run’s data.

What SOPs need to be in place, and what efforts are going toward filling that need?

Elkins: Labs will need to validate the new instrument and kits. An SOP will need to be written to designate the sample preparation and handling process prior to sequencing, as well as the data analysis scheme, thresholds, reporting, and data storage following sequencing. There are manufacturer protocols and peer-reviewed publications that labs can draw from in conducting their internal studies and writing their SOPs.

Describe a case that exemplifies the unique and advanced nature of NGS.

Elkins: There have now been many cases that have been solved using NGS especially with investigative genetic genealogy. The Golden State Killer case is still probably the most well-known. Others include a Dutch criminal offender case, 1990 Labor Day murders, and the sexual assault and murder cases of Tanya Van Cuylenborg and Jay Cook — but there are many more.

What’s next for NGS?

Elkins: NGS will be applied more routinely by forensic labs to solve more cold cases that have been on their dockets for years. Law enforcement and scientists have been hoping for years that the technology would be developed in their lifetimes so they can finally solve them. NGS will also be applied to low-quantity samples in current casework so that scientists can get the most information from limited samples to provide investigators and law enforcement sufficient data to solve the cases.

What do crime scene investigators need to know about NGS?

Elkins: NGS is an extremely sensitive tool and avoiding contamination in sample collection is of the utmost importance and key to correct analysis. The power of NGS is going to be a benefit to your investigations.

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